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Introduction

The SuperScript™ Indirect cDNA Labeling System is a highly efficient system for generating fluorescently labeled cDNA for use on microarrays in gene expression studies (De Risi et al., 1996; Eisen & Brown, 1999). It uses an aminoallyl-modified nucleotide and an aminohexyl-modified nucleotide together with other dNTPs in a cDNA synthesis reaction with SuperScript™ III Reverse Transcriptase. After a purification step to remove unincorporated nucleotides, the amino-modified cDNA is coupled with a monoreactive, N-hydroxysuccinimide (NHS)-ester fluorescent dye. A final purification step removes any unreacted dye, and the fluorescently labeled cDNA is ready for hybridization to microarrays.

This system uses 5–20 μg of total RNA or 0.4–2 μg of mRNA as starting material, and is compatible with Alexa Fluor 555 and Alexa Fluor 647 fluorescent dyes from Invitrogen, Cy3 and Cy5 dyes from Amersham Biosciences, or other monoreactive NHS-ester dyes from a variety of manufacturers.

Advantages of the System

SuperScript™ III Reverse Transcriptase in the first-strand synthesis reaction ensures high specificity and high yields of cDNA, as well as more full-length cDNA

Use of two amino-modified nucleotides in the cDNA synthesis reaction results in a greater incorporation of fluorescent dye and higher signal intensity with small amounts of starting material

Unbiased incorporation of amino-modified dNTPs and the high efficiency of the coupling reaction result in an even distribution of fluorescent signal and high overall levels of fluorescence, increasing the sensitivity and reproducibility of array hybridizations

System includes all major reagents and materials for preparing fluorescently labeled cDNA, except fluorescent dye

Advantages of SuperScript™ III Reverse Transcriptase

SuperScript™ III Reverse Transcriptase is an engineered version of M-MLV RT with reduced RNase H activity and increased thermal stability. The enzyme can be used to synthesize first-strand cDNA from total RNA or mRNA at temperatures up to 55ºC, providing increased specificity, higher yields of cDNA, and more full-length product than other reverse transcriptases. The SuperScript™ III RT in this kit is provided at an optimal concentration and used at an optimal temperature for incorporating amino-modified nucleotides in first-strand cDNA synthesis.

Anchored Oligo(dT)20

Anchored oligo(dT)20 primer is a mixture of 12 primers, each consisting of a string of 20 deoxythymidylic acid (dT) residues followed by two additional nucleotides represented by VN, where:

V is dA, dC, or dG

N is dA, dC, dG or dT

The VN “anchor” allows the primer to anneal only at the 5′ end of the poly(A) tail of mRNA, providing more efficient cDNA synthesis for labeling applications. Dye Compatibility This kit has been developed using Alexa Fluor fluorescent dyes from Invitrogen and CyDye™ fluorescent dyes from Amersham Biosciences.

Other monofunctional, N-hydroxysuccinimide (NHS)-reactive fluorescent dyes are compatible with this system.

Materials Supplied by the User

In addition to the kit components, you should have the following items on hand before using the SuperScript™ Indirect cDNA Labeling System.

Monofunctional, NHS-reactive fluorescent dye

Vortex mixer

Microcentrifuge

Aerosol resistant pipette tips

Water baths or incubator

1 N NaOH

1 N HCl

Sterile microcentrifuge tubes

100% Isopropanol

100% Ethanol

75% Ethanol

Control Reaction

We recommend performing the labeling procedure using the Control HeLa RNA included in the system to determine the efficiency of the labeling reaction. The section on First-Strand cDNA Synthesis describes how to set up the control reaction.

Isolating RNA

Introduction

High-quality, intact RNA is essential for full-length, high-quality cDNA synthesis. In this step, you isolate total RNA or mRNA using a method of choice.

The quality of the RNA is critical for successful labeling and hybridization. The presence of contaminants in the RNA may significantly increase background fluorescence in your microarrays. Carefully follow the recommendations below to prevent RNase contamination.

General Handling of RNA

When working with RNA:

Use disposable, individually wrapped, sterile plasticware.

Use aerosol resistant pipette tips for all procedures.

Use only sterile, new pipette tips and microcentrifuge tubes.

Wear latex gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin.

Use proper microbiological aseptic technique when working with RNA.

Dedicate a separate set of pipettes, buffers, and enzymes for RNA work.

Microcentrifuge tubes can be taken from an unopened box, autoclaved, and used for all RNA work. RNase-free microcentrifuge tubes are available from several suppliers. If it is necessary to decontaminate untreated tubes, soak the tubes overnight in a 0.01% (v/v) aqueous solution of diethylpyrocarbonate (DEPCtreated), rinse the tubes with sterile distilled water, and autoclave the tubes.

You can use RNase AWAY™ Reagent, a non-toxic solution available from Invitrogen to remove RNase contamination from surfaces. For further information on controlling RNase contamination, see Ausubel, et al. and Sambrook, et al (Ausubel et al., 1994; Sambrook et al., 1989).

Isolating RNA

This system is designed for use with 5–20 μg total RNA or 0.4–2 μg of mRNA. To isolate total RNA, we recommend the PureLink™ Micro-to-Midi Total RNA Purification System, TRIzol Reagent, or (for high-throughput applications) the PureLink™ 96 RNA Purification System. To isolate mRNA, we recommend the FastTrack 2.0 mRNA Isolation Kits or the FastTrack MAG mRNA Isolation Kits. (see Ordering information). After you have isolated the RNA, check the quality of your RNA preparation as described below.

Checking the RNA Quality

To check RNA quality, analyze 500 ng of RNA by agarose/ethidium bromide gel electrophoresis. You can use a regular 1% agarose gel or a denaturing agarose gel (Ausubel et al., 1994). For total human RNA using a regular agarose gel, mRNA will appear as a smear from 0.5 to 9 kb, and 28S and 18S rRNA will appear as bands at 4.5 kb and 1.9 kb, respectively. The 28S band should be twice the intensity of the 18S band. If you are using a denaturing gel, the rRNA bands should be very clear and sharp. If you do not load enough RNA, the 28S band may appear to be diffuse. A smear of RNA or a lower intensity 28S band with an accumulation of low molecular weight RNA on the gel are indications that the RNA may be degraded, which will decrease the labeling efficiency. If you do not detect any RNA, you will need to repeat RNA isolation. Refer to the Troubleshooting section.

Storing RNA After preparing the RNA, we recommend that you proceed directly to First-Strand cDNA Synthesis. Otherwise, store the RNA at –80ºC.

First-Strand cDNA Synthesis

Introduction After you have isolated RNA and checked the quality of your RNA preparation, you are ready to synthesize cDNA. Before Starting The following materials are supplied by the user:

5–20 μg total RNA or 0.4–2 μg mRNA

1 N NaOH

1 N HCl

Water baths, heating block, or incubator set at 46ºC and 70ºC

Ice

0.5-ml or 1.5-ml RNase-free microcentrifuge tubes

The following materials are supplied in the kit:

Anchored Oligo(dT)20 primer

Random hexamers (for mRNA starting material only)

dNTP mix, including amino-modified nucleotides

5X First-Strand buffer

0.1 M DTT

RNaseOUT™

SuperScript™ III RT

DEPC-treated water

Control HeLa RNA, optional

3 M Sodium Acetate, pH 5.2

Control HeLa RNA Control HeLa RNA is included in the kit to help you determine the efficiency of the labeling procedure. We strongly recommend that you perform the control reaction if you are a first-time user of the SuperScript™ Indirect cDNA Labeling System. Instructions are provided on the next page to set up separate cDNA synthesis reactions for your sample and the Control HeLa RNA. Equations for calculating the amount of coupled dye in the control reaction are provided below. RNaseOUT™ Recombinant RNase Inhibitor has been included in the system to safeguard against degradation of target RNA due to ribonuclease contamination of the RNA preparation.

First-Strand cDNA Synthesis Reaction The following procedure is designed to convert 5–20 μg of total RNA or 0.4–2 μg of mRNA into first-strand cDNA.

Note: If you are setting up a control reaction (recommended for first-time users), use 10 μl of the Control HeLa RNA (1 μg/μl) supplied in the kit.

Mix and briefly centrifuge each component before use.

Prepare reaction(s) as follows, using 0.5-ml or 1.5-ml RNase-free tubes:

Component

Sample

Amount

5–20 μg total RNA or 0.4–2 μg mRNA

X μl

--

Control HeLa RNA (1 μg/μl)

--

10 μl

Anchored Oligo(dT)20 Primer (2.5 μg/μl)

2 μl

2 μl

Random hexamers (only if using mRNA)

1 μl *

--

DEPC-treated water

to 18 μl

to 18 μ

*For mRNA, use both anchored oligo(dT)20 and random hexamers. For total RNA, use only 2 μl of anchored oligo(dT)20.

Incubate tubes at 70ºC for 5 minutes, and then place on ice for at least 1 minute.

Add the following to each tube (sample and control) on ice:

Component

Volume

5X First-Strand buffer

6 μl

0.1 M DTT

1.5 μl

dNTP mix (including amino-modified nucleotides)

1.5 μl

RNaseOUT™ (40 U/μl)

1 μl

SuperScript™ III RT (400 U/μl)

2 μl

Final Volume

30 μl

Mix gently and collect the contents of each tube by brief centrifugation. Incubate tube at 46°C for 2–3 hours. Note: A 3-hour incubation results in 20–30% higher cDNA yield than a 2-hour incubation.

After incubation, proceed directly to Alkaline Hydrolysis and Neutralization, below.

Hydrolysis and Neutralization After cDNA synthesis, above, immediately perform the following hydrolysis reaction to degrade the original RNA:

Purifying First-Strand cDNA

Introduction After you have generated cDNA with amino-modified nucleotides, you need to purify the cDNA to remove unincorporated dNTPs and hydrolyzed RNA.

Catalog Numbers L1014-01 and L1014-02 include a Purification Module developed for use with the system. Use the S.N.A.P.™ Column Purification procedure below to purify your amino-modified cDNA using this Purification Module.

Catalog Number L1014-03 does not included a Purification Module. Use your preferred method of purification instead of the S.N.A.P.™ Column Purification procedure, and proceed to the Ethanol Precipitation procedure below. You must perform the Ethanol Precipitation step even if you are using your own purification procedure.

Before Starting The following materials are supplied by the user:

Microcentrifuge

1.5-ml microcentrifuge tube

100% Ethanol

75% Ethanol

The following materials are supplied in the Core Module:

2X Coupling Buffer

3 M Sodium Acetate, pH 5.2

Glycogen (20 mg/ml)

The following materials are supplied in the Purification Module (Catalog Numbers L1014-01 and L1014-02):

DEPC-treated water

S.N.A.P.™ column(s) and clear collection tube(s)

Loading Buffer plus isopropanol

Wash Buffer plus ethanol

The pellet should be completely dry at the end of the purification procedure to ensure complete removal of the ethanol. The presence of ethanol can inhibit the labeling reaction.

S.N.A.P.™ Column Purification Use the following procedure to purify the cDNA using the components of the Purification Module (Cat. nos. L1014-01 and L1014-02). If you are using Cat. No. L1014-03, purify using your method of choice and then proceed to Ethanol Precipitation below.

Note: Before starting the procedure, be sure to add isopropanol to the Loading Buffer and ethanol to the Wash Buffer.

Add 300 μl of ice-cold 100% ethanol, and incubate the tube at –20ºC for at least 30 minutes. Note: You can incubate the tube overnight if desired.

Spin the tube at 14,000 × g at 4ºC for 10–20 minutes. Carefully remove and discard the supernatant. Note: You can spin at room temperature if a refrigerated centrifuge is unavailable; however, the yield may be slightly less.

The sample can stored at –20º C for up to one week prior to hybridization. Avoid freeze/thawing. To determine the efficiency of the labeling reaction, proceed to Assessing Labeling Efficiency.

Hybridization After purification, you are ready to use the labeled cDNA in any application of choice, including glass microarray hybridization. Follow the preparation and hybridization instructions for your specific application.

Troubleshooting

Problem: 28S and 18S bands are not observed after isolation of total RNA and agarose gel electrophoresis

Cause

Solution

Too little RNA loaded on the gel

Be sure to load at least 250 ng of RNA for analysis.

RNA is degraded due to RNase activity

Follow the guidelines to avoid RNase contamination. Use a fresh sample for RNA isolation.

Problem: Yield of cDNA from the first-strand synthesis reaction is low

Cause

Solution

Temperature too high during cDNA synthesis

Perform the cDNA synthesis at 46ºC.

Incorrect reaction conditions used

Verify that all reaction components are included in the reaction and use reagents provided in the system. Verify the reaction conditions using the control RNA provided in the kit.

Concentration of template RNA is too low

Increase the concentration of template RNA. Use at least 5 μg of total RNA or 0.4 μg of mRNA.

Poor quality RNA used or RNA is degraded

Check the quality of your RNA preparation. If RNA is degraded, use fresh RNA.

RNase contamination

Use the RNaseOUT™ included in the kit to prevent RNA degradation.

RT inhibitors are present in your RNA sample

Inhibitors of RT include SDS, EDTA, guanidinium chloride, formamide, sodium phosphate and spermidine (Gerard, 1994). Remove inhibitors from your RNA sample by performing an additional 70% ethanol wash after ethanol precipitation during RNA isolation and purification. Test for the presence of inhibitors by mixing 1 μg of control RNA with 25 μg total RNA or 1 μg mRNA and compare the yields of first-strand synthesis.

Improper storage of SuperScript™ III RT

Store the enzyme at –20ºC.

Problem: Yield of labeled cDNA from the control reaction is low

Cause

Solution

cDNA has been lost in the purification step following cDNA synthesis

Follow the S.N.A.P.™ Column purification procedure and ethanol precipitation procedure without modifications. Overnight ethanol precipitation may result in higher yields.

cDNA has been lost in the purification step after labeling

Measure the amount of labeled cDNA in the control reaction before and after purification. Follow the purification procedure without modifications.

Problem: Amount of coupled dye in the control reaction is low (< 40 pmoles) and/or fluorescence of labeled cDNA is low

Cause

Solution

Reaction tubes have been exposed to light

Avoid direct exposure of the labeling reaction to light. Use an amber tube for collection of the final product.

Dye solution has been exposed to light

Repeat labeling reaction with fresh mixture of dye, being careful to avoid direct exposure to light.

DMSO used to prepare dye mixture was contaminated with water.

Prepare a new mixture of dye using fresh DMSO. Carefully follow the instructions for storing and handling DMSO